Oncoretroviral expression is transcriptionally silenced in embryonic and hematopoietic stem cells (HSCs). This is associated with methylation of viral and internal promoters. We determined whether self-inactivating (SIN) lentiviral vectors (LV) would circumvent proviral silencing in HSCs. We studied long-term expression, methylation, and position effects (PE) from two GFP-encoding SIN-LV containing erythroid enhancers and the human ankyrin-1 promoter (h-Ank-P) using the murine secondary bone marrow (BM) transplant assay. Proviral expression was detected in RBC 6-11 months following transplant only in 28 of 49 secondary mice, with 0.9 ± 0.2 copy/cell and oligoclonally integrated provirus in BM, spleen, and thymus. Twenty-one of 49 secondary mice lacked integrated provirus. Secondary mice containing provirus also had GFP-expressing RBCs, although proviral copy number did not always correlate with expression, suggesting either proviral methylation or chromatin PE. The endogenous h-Ank-P was partially methylated in nonerythroid cell lines and unmethylated in erythroid cell lines. However, h-Ank-P in the provirus was unmethylated in erythroid and nonerythroid cells within secondary murine BM. Despite lack of methylation, GFP expression was variable in secondary BFU-e and in single-copy mouse erythroleukemia cell clones. Taken together, these data show that erythroid-specific SIN-LV express long term and resist methylation-associated proviral silencing, but may require additional elements to confer position-independent expression.
ASJC Scopus subject areas
- Molecular Biology